The overall objective of this project is elucidate the mechanism of action of the enzyme choline acetyltransferase in vitro, and to determine to what extent these studies relate to the behavior of the enzyme, in vivo. Improved purification of the enzyme from human placenta by the use of affinity chromatography and physical characterization of the enzyme including determination of subunit structure will be pursued. In addition, kinetic studies on the mechanism of action and the effect of salts on enzyme activity will be conducted by measuring isotope exchange at equilibrium and solvent deuterium isotope effects. The in vitro properties of the enzyme will be compared to the properties of the enzyme in situ in hopes of more closely approximating in vivo conditions. These latter studies will involve the use of permeabilized synaptosomes as a vehicle for retaining the enzyme within a macromolecular environment approximating that found in vivo. This system will enable pH, ionic strength, and substrate concentration to be varied, while maintaining the enzyme within the synaptosome. It is anticipated that the combination of in vitro and in situ studies outlined in this proposal will provide a better understanding of the kinetic behavior of choline acetyltransferase, and hence lead to a better understanding of the mechanisms of acetylcholine synthesis in vivo.
